The major findings of our HTLV-I studies have been: 1) Increased HTLV-I specific CD8+ cells have been shown to be elevated in the peripheral blood and CSF of HAM/TSP patients and directly proportional to the amount of HTLV-I proviral DNA and RNA. These antigen-specific T cells are considered to be immunopathogenic and may be directly involved in virus-host interactions in the CNS. We have extended these observations to explore antigen-specific T cell responses to other viruses including EBV, CMV and HHV6 using pools of select peptides. We are extending these observations to define the T cell receptor repertoire in the peripheral blood and CSF of HAM/TSP and MS patients and have demonstrated T cell clonal expansions particularly in the CSF of MS patients. We have also begun to explore the B cell receptor repertoire from single cells in the CSF of these patients and have successfully expressed paired Vh and Vl chains. The specificity of these antibodies are being investigated. 2) We continue to explore the role of monocytes in immune regulation and disease progression in patients with HTLV-1-associated inflammatory diseases. We detected HTLV-1 DNA in monocyte subsets (classical, intermediate, and nonclassical) and found that infection impacts surface receptor expression, migratory function, and subset frequency. The frequency of nonclassical patrolling monocytes is increased in HTLV-1-infected individuals, and they have increased expression of CCR1, CXCR3, and CX3CR1. The viral DNA level in nonclassical monocytes correlated with the viral DNA level in CD4(+) and CD8(+) T cells. Altogether, these data suggest an increased recruitment of classical monocytes to inflammation sites that may result in virus acquisition and, in turn, facilitate virus dissemination and viral persistence. Our findings thus provide new insight into the importance of monocyte infection in viral spread and suggest targeting of monocytes for therapeutic intervention. 3) CD4+CD25+ regulatory T cells are important in the maintenance of immunological self-tolerance and in the prevention of autoimmune diseases. We have demonstrated that in HTLV-I infected CD4+CD25+ T cells of patients with HAM/TSP, the regulatory T cells were dysfunctional and lower than that of healthy individuals. Using a recently developed nanotrap technology, we have begun to examine the role of exosomes from HAM/TSP and have shown that these vesicles contain HTLV-I tax protein in CSF of patients. HTLV-I tax containing exosomes were also shown to reduce the suppressive capacity of Treg cells. 4) Basic laboratory investigations have demonstrated the importance of the cytokine IL-15 in the life and death of lymphocytes and for its role in autoimmune disorders such as HAM/TSP. IL-15 is pivotally involved in the survival of CD8+ memory T-cells including self-directed cells and we have shown that in HAM/TSP, IL-15 is essential for the survival of HLA class I restricted virus antigen-specific effector and memory CD8+ T-cells. These CD8+ antigen-specific CTL are thought to play a major role in the immunopathogenesis of HAM/TSP since they have been localized in brain and spinal cord sections of patients. As IL-15 is a pro-inflammatory cytokine that stimulates the production of inflammatory cytokines, the release of IL-15 induced by HTLV-I tax in patients with HAM/TSP may underlie the pathogenesis of this autoimmune disease. The mode of action of IL-15 and its receptor subunits IL-15 and IL-15R alpha are coordinately stimulated and expressed following HTLV-I tax stimulation. We have shown that this trans-stimulation can be virtually totally inhibited for NK and CD8+ T-cells by the addition of a humanized monoclonal antibody, Hu MiKbeta1, that blocks IL-15 binding to the IL-2/IL15Rbeta; receptor subunit expressed on these cells. Collaborative research by the Viral Immunology Section, NINDS and the Metabolism Branch, NCI have initiated a phase I clinical trial for the treatment of HAM/TSP using Hu MiK-beta-1 that blocks the action of IL-15. 5) This understanding of the roles of IL-2/IL-15 in the pathogenesis of HAM/TSP has led to targeted therapeutic approaches. Additionally, given the fact that IL-2/IL-15 signal through the common gamma chain receptor, a novel potentially more effective targeted therapeutic approach to HAM/TSP might consist of blocking the common gamma chain with an antagonizing peptide. This would inhibit the action of multiple cytokines implicated in the disease and might be better tolerated than monoclonal antibodies or small molecule inhibitors. A peptide called BNZ gamma is a novel anti-cytokine peptide that was designed based on the presence of a moderately conserved region among all the cytokines sharing the common gamma chain receptor and was found to selectively block signaling of IL-2, IL-15, and IL-9. We have examined the effects of BNZ-g; on multiple in vitro markers of immune activation in HAM/TSP. The peptide significantly suppressed spontaneous proliferation (SP) of the PBMCs, reduced the frequency of Tax-specific CD8+ T-cells, and the degree of STAT5 phosphorylation after 20h of culture. It was as effective as a combination of monoclonal antibodies against CD25 and CD122 in reducing SP and STAT5 phosphorylation. These observations have led to a phase I clinical trial on the safety and tolerability of BNZ-g in healthy volunteers. 6) We are continuing to assess virus-specific immune responses from MS patients and controls using virus-peptide pools and can demonstrate EBV-specific CD4+ and CD8+ T cells that can be detected by HLA-restricted tetramers. In addition, we are using our newly developed digital based PCR methodology (ddPCR) for the detection of human viruses. We have characterized PCR primer and pair probes for the detection of HTLV-I, HTLV-II, EBV, JCV, CMV and HHV6 A and HHV6B. We have multiplexed this system in which we can now simultaneously detect and quantify herepesvirus sequences in human material. We have quantitated CMV, HHV-6A, HHV-6B and EBV viral DNA in human astrocytomas (WHO grade I-IV) and non-neurological disease control tissues and have demonstrated HHV-6B and EBV, but not CMV from high-grade astrocytomas. We are also assessing novel anti-herpesvirus small molecule compounds for their in vitro anti-viral effects. 7) We continue to extend our work on the detection of the human herpesvirus (HHV-6) from brain resections of patients with mesial temporal lobe epilepsy, patients with neurologic complications following allogeneic bone marrow transplants, and patients with astrocytomas. We have developed a novel electrochemiluminescent ELISA method for the quantitative detection of antibodies to HHV-6 IgG. We have screened large panels of sera from patients with MS, encephalitis and controls. Moreover, we have begun to explore the role of these viruses in pediatric MS samples from University of California San Francisco and established a collaboration with Childrens Hospital DC to obtain pediatric saliva and serum from children with seizures and fever. From MS tissue, we have extended our initial PET studies of the 18 kDa translocator protein (TSPO) that is strongly unregulated in CNS injury and thus of interest as a biomarker of neuroinflammation. Through multi-color immunofluorescence immunohistochemistry we can localize TSPO expression to areas of MS lesions and microglial activation. We are also using a flow based analysis to determine if TSPO can be used as a biomarker from peripheral blood of MS patients and controls.